Composition, formulae, devices and methods for control of specificity and inclusivity of microorganisms containing closely related antigen epitopes

ABSTRACT

Compositions, formulae, devices and methods for the detection of target microorganisms, such as by visual immunoprecipitate assay, enzyme linked immunoassay, chemiluminescence, immunoblotting, or similar detection technology, wherein detection requires the discrimination among closely related genera, species and strains of antigenically related microorganisms based on immunological reactivity of a highly conserved antigen epitopes with a reagent system comprised of an antibody linked to a detecting reagent. The invention permits a detectable event to occur by exposing inaccessible but highly conserved and specific antigen epitopes to the detecting reagent. Exposure of such antigen epitopes without inactivating microbial metabolism allows for specific detection.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application is a continuation of U.S. patent applicationSer. No. 09/374,560, filed Aug. 13, 1999, now allowed; which applicationclaims priority from U.S. Provisional Application No. 60,096,566, filedAug. 14, 1998; which applications are incorporated herein by referencein their entirety.

TECHNICAL FIELD

[0002] The present invention relates generally to compositions,formulae, devices, and methods for detecting and identifyingmicroorganisms and, more particularly, to compositions, formulae,devices, and methods for detecting microorganisms by exposing highlyconserved antigenic sites of target microorganisms without inactivatingthe microorganisms' ability to grow to sufficient levels to be detectedby an antibody linked detection system.

BACKGROUND OF THE INVENTION

[0003] Microbial diseases have long been a major health concernworldwide. Significant increase in the frequency and severity ofoutbreaks have occurred throughout the world. New pathogenic bacteria,such as E. coli 0157:H7, have been identified. Furthermore, previouslyrecognized pathogenic genera have mutated to form drug resistant highlyinfectious strains such as Salmonella typhimirium DT 104. A key featurein the prevention of such diseases is early diagnosis. Epidemiologistsmust look for microbial contamination in the environment as well as infood products to find the effective disease prevention strategies.

[0004] One example is the outbreak in 1992 of Enterohemorrhagic E. coli(EHEC) in the Pacific Northwest of the United States due to contaminatedground beef. EHEC is a relatively “newly discovered” pathogen. EHEC wasfirst isolated in 1975, and it was not until 1982 that E. coli 0157:H7was associated with two food related outbreaks of hemorrhagic colitis inthe United States. The reported incidence of E. coli 0157:H7 cases isincreasing. Typically, E. coli strains are harmless commensals, but afew strains are pathogenic. EHEC is particularly virulent and cantrigger deadly complications, including severe abdominal cramps andacute renal failure in children as well as cardiovascular and centralnervous system problems.

[0005] As another example, Salmonella is the leading cause (more than50%) of total bacterial foodborne disease outbreaks, according to theUnited States Centers for Disease Control (CDC) surveillance of foodbomediseases. More than 40,000 cases per year were reported to the CDCduring the period 1988-1992. Salmonella can infect a broad variety ofwarm- and cold blooded animals, and can survive for long periods of timeoutside a host.

[0006] In a further example, Salmonella typhimurium DT 104 was firstidentified in the United Kingdom in the early 1990s. It is a highlyadapted drug resistant strain of Salmonella known for its virulence.Resultingly, significant clinical interest has surrounded this serotype.S. typhimurium DT 104 contains core cell wall antigen epitopes that arehighly conserved among the genus Salmonella.

[0007] Listeria, a genus of gram positive bacteria, is widelydistributed in nature, having been isolated from soil, water, vegetationand many animal species. The detection frequency for Listeria in theagricultural environment appears to be increasing. For specificoutbreaks of listeriosis, estimates place mortality at 30% to 40% ofaffected patients, however, little is known of the minimum infectivedose. One particularly troublesome aspect of Listeria control in foodsis that Listeria can grow at temperatures as low as −0.4° C. and as highas 44° C. These factors all contribute to the increasing significance ofListeria as a food pathogen.

[0008]Campylobacter jejuni and coli have recently been identified as thelead causes of enteritis, especially from poultry sources. This has ledto an increased need to discriminate these two species from severalother Campylobacter species which are not human pathogens. This requiresthe differential selection of more specific cell wall membrane antigenepitopes.

[0009] The ability to monitor potential environmental and food sourcesof microbial contamination quickly and easily, but with very highspecificity, would reduce the risk of human infection. Therefore, ananalytical method which affords high specificity, preferably combinedwith a device able to assay for microorganisms, including bacteria,yeasts, molds, fungi, parasites and viruses, that requires no special ortechnical equipment, can be performed in the field and does not requirespecial skills would be useful. In the case of foodborne bacterialcontamination, four of the major disease-related organisms areSalmonella, Listeria, EHEC and Campylobacter.

[0010] While there are a number of Salmonella, Listeria, and EHECdetection methods presently available, trained laboratory techniciansand a minimum of 2-5 days are required to obtain test results by thestandard cultural methods of analysis. New, more rapid methods are basedon such techniques as enzyme linked immunoassay (EIA), DNAhybridization, immunodiffusion, or growth/metabolism measurements. Whiletaking much less time than the cultural methods, these rapid tests stillrequire skilled technical training, a functional laboratory, andspecialized equipment. These tests generally take a total of two or moredays, including considerable hands-on time. Campylobacter detectionmethodology to date is technically intensive requiring fastidious mediaand environmental conditions, in addition to well-trained analysts.

[0011] Another recent technology in the diagnostic field involveslateral flow immunoassays. Such tests have been developed for thedetection of human chorionic gonadotropin (hCG), and applied topregnancy testing. Typically, a monoclonal or polyclonal antibody isimmobilized in a discrete band near the distal end of a solid carrierstrip, called the detection zone. Another amount of antibody is labeledwith a detection reagent such as an inorganic sol or dyed polystyreneparticle. This labeled antibody is reversibly fixed near the proximalend of the carrier strip. Upon hydration of the proximal end with asample fluid potentially containing the antigen, the antigen reacts withthe labeled antibody and the complex passes through the zone ofimmobilized antibody, forming a sandwich upon reacting with theimmobilized antibody. The capture of the chromogenic reagent-antigencomplex causes the formation of a visible signal in the detection zone.

[0012] Two major challenges must be addressed to distinguish pathogenicbacteria, as opposed to distinguishing hormones or other solublemolecular targets. These challenges are the need to detect all of thestrains of a pathogenic microorganism in the presence of numerousantigenically related organisms, with a low tolerance for false positiveresults and a very low, preferably zero, tolerance for false negatives.The second challenge is the physical size and heterogeneity of themicroorganism itself. A typical clinical diagnostic test, such as a testfor hCG in urine, is focused on detecting a single, small, unique entity(i.e., a hormone) in a well characterized matrix (e.g., urine).Furthermore, the structure of the analyte (hCG) is defined and uniformin size and composition.

[0013] Pathogen detection, for example, a test for Salmonella, mustdistinguish a particular pathogenic strain from nonpathogenic strains ofsimilar microorganisms, such as Citrobacter spp. and Enterobacter spp.In contrast to the well-defined small size and structure of mosthormones or marker proteins, microorganisms are very large, theirsurfaces are heterogeneous containing many distinct antigen epitopesthat can undergo changes, such as the phase-switching of Salmonellaflagella.

[0014] In addition, the cell wall membrane of many microorganismscontain antigen epitopes, such as lipopolysaccharides, which arerepeated with a high degree of consistency within a given genus. Theseantigen epitopes serve as highly desirable targets for reaction withcomplimentary specific antibodies which, in turn, provides a method ofhigh accuracy with low false positives. The ability to isolate and bindto antibodies reacting with these highly conserved antigen epitopes isdifficult, however, because they can be sterically hindered by O-antigenpolysaccharide chains. They are generally inaccessible because of aphenomenon known as steric interference. This steric interference isprovided by the surface antigen epitopes of the microorganism. Examplesof surface structures known to contribute to this interference aresurface proteins, group specific lypopolysaccharides, flagella, andcellular encapsulation.

[0015] Although, aggressive treatments are available which will exposeinterior antigen epitopes, these treatments destroy cell viability andin many cases disrupt cellular integrity completely. Examples of suchtreatments are heat treatment (boiling or autoclaving) and chemicalextraction (nitrous acid digestion). The significant shortcoming ofthese extractions is that they result in death of the microorganism.Therefore, if the cell population had not reached a sufficientlydetectable level prior to inactivation, a negative determination willresult.

[0016] Thus, there is a need in the art for methodologies that willallow the simultaneous exposure of highly conserved masked antigenepitopes while still allowing the microorganisms to multiply. Further,there is a need in the art to incorporate improved selectivity forhighly conserved target antigen epitopes of specific species in apopulation of heterogeneous microorganisms in a variety of matrices. Thepresent invention provides these and other, related advantages.

SUMMARY OF THE INVENTION

[0017] The present invention generally provides a novel, antigenicepitope exposing microorganism growth composition. In one aspect, theinvention provides a composition comprising a general enrichment mediaand at least one structure modifying organic chemical. In oneembodiment, the structure modifying organic chemical is2,4-dinitrophenol or carbonyl cyanide-m-chlorophenyl hydrazone. Inanother embodiment, the structure modifying organic chemical is2,4-dinitrophenol. In yet another embodiment, the general enrichmentmedia is selected from a variety of readily made or commerciallyavailable media including Terrific Broth, SOB medium, SOC medium, LBmedium, NZCYM medium, minimal medium, lactose broth, buffered peptonewater, Brain Heart Infusion medium, Haemophilus broth, tryptic soybroth, and nutrient broth.

[0018] It is another aspect of the present invention to provide a methodfor detecting a microorganism in a test sample by contacting the testsample with a composition comprising general enrichment media and atleast one structure modifying organic chemical, thereby forming amixture. This mixture is then incubated for a time sufficient to allowfor detectable levels of microorganisms to develop, after which thepresence of specific microorganisms is detected. In one embodiment ofthis aspect of the invention the mixture is contacted with a detergentprior to or contemporaneous with detection. In another embodiment, themixture is contacted with a detergent and heated prior to orcontemporaneous with detection. In one embodiment, the detergent isanionic. In yet another embodiment, the detergent is non-ionic. Incertain embodiments, the anionic detergent may be selected from sodiumdodecyl sulfate and sodium deoxycholate. In certain embodiments, thenon-ionic detergent is NP-40, tergitol, or triton X-100. In certainother embodiments, the mixture is heated in the presence of thedetergent to a temperature, between 40° C. and 121° C.

[0019] In certain embodiments of the detection method, the microorganismdetected is Listeria, Enterohemorrhagic E. coli, Salmonella, orCampylobacter.

[0020] Turning to another aspect of the invention, a method is providedfor detecting the presence of Listeria, Enterohemorrhagic E. coli,Salmonella, or Campylobacter in a test sample wherein the test sample iscontacted with a composition comprising general enrichment media and atleast one structure modifying organic chemical, followed by incubationof this mixture for a time sufficient to allow for detectable levels ofmicroorganisms to develop. Subsequent to the development of detectablelevels of microorganisms in the mixture, the presence of Listeria,Enterohemorrhagic E. coli, Salmonella, or Campylobacter is specificallydetected. In one embodiment of this aspect of the invention the mixtureis contacted with a detergent prior to or contemporaneous withdetection. In another embodiment, the mixture is contacted with adetergent and heated prior to or contemporaneous with detection. In oneembodiment, the detergent is anionic. In yet another embodiment, thedetergent is non-ionic. In certain embodiments, the anionic detergentmay be sodium dodecyl sulfate or sodium deoxycholate. In certainembodiments, the non-ionic detergent is NP-40, tergitol, or tritonX-100. In certain other embodiments, the mixture is heated in thepresence of the detergent to a temperature, between 40° C. and 121° C.

[0021] In another embodiment, the detection methodologies describedherein utilize an immunoassay. In certain embodiments, the immunoassayis selected from a visual immunoprecipitate assay, an enzyme linkedimmunoassay, chemiluminescence, and immunoblotting. In certain otherembodiments, the immunoassay is a visual immunoprecipitate assay. Alsoprovided in certain embodiments are immunoassays which utilize acomplementary monoclonal antibody, polyclonal antibody, or an antibodyfragment, wherein said antibody or antibody fragment is specific for ahighly conserved cell wall epitope in the target microorganism.

[0022] In another aspect of the invention, a method is provided,comprising contacting a test sample containing a microorganism with animmunoaffinity based detection device, wherein the test sample has beenpreviously propagated in the presence of a structure modifying organicchemical.

[0023] The invention also provides a method for propagating amicroorganism such that cell wall antigen epitopes of the microorganismare altered by contacting a test sample with a composition comprisinggeneral enrichment media and at least one structure modifying organicchemical, and propagating the microorganism therein.

[0024] Turning to yet another aspect of the invention, a method fordetecting microorganism specific epitopes on a target microorganism in atest sample is provided, comprising propagating a microorganism in atest sample in a permissive general enrichment media, wherein said mediacomprises a structure modifying organic chemical, and contacting thetest sample with a microorganism specific antibody linked to a detectingreagent, wherein reaction with the antibody indicates the presence ofthe microorganism. In further embodiment, contact between the testsample and the antibody occurs in device or assay system. In yet anotherembodiment, the assay system is selected from a visual immunoprecipitateassay, an enzyme linked immunoassay, chemiluminescence, andimmunoblotting. In another embodiment, the assay device is a lateralflow detection device. In certain embodiments, the antibody used in theabove methods is specific for a microorganism selected from Salmonella,Enterohemorrhagic E. coli, Listeria, and Campylobacter.

[0025] It is another aspect of the present invention to provide alateral flow device for detecting a target microorganism in a samplecomprising a microorganism specific antibody and a test samplepreviously propagated in a general enrichment media, the mediacomprising at least one structure modifying organic chemical. In anotherembodiment of this aspect of the invention the antibody is specific forany one of Salmonella, Enterohemorrhagic E. coli, Listeria., orCampylobacter.

[0026] These and other aspects of the present invention will becomeevident upon reference to the following detailed description andexamples. In addition, the various references set forth below describein more detail certain procedures or compositions (e.g., antibodies,detection methodologies, etc.), and are therefore each incorporatedherein, by reference, in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

[0027] Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

[0028] The term “antibody” as used herein includes polyclonal,monoclonal, humanized, chimeric, and anti-idiotypic antibodies, as wellas fragments thereof such as F(ab′)₂ and Fab fragments and otherrecombinantly produced binding partners. Further, the antibodies may becovalently linked to or recombinantly fused to an enzyme, such asalkaline phosphatase, horse radish peroxidase, α-galactosidase, and thelike.

[0029] “Structure modifying organic chemical” refers to an organicchemical capable of altering the composition of the cell wall of amicroorganism, such that specific and conserved ligands are exposed.Briefly, such organic chemicals typically inhibit the transfer ofsterically interfering epitopes to the cell wall. Such organic chemicalsinclude, but are not limited to, 2,4-dinitrophenol, carbonylcyanide-m-chlorophenyl hydrazone or similar electron uncouplers (i.e.,disabling proton motive force), which have the effect of exposing highaffinity and specific epitopes which are recognized by monoclonal orpolyclonal antibodies.

[0030] The term “general enrichment media” refers to any media which isknown to be useful for facilitating the growth of microorganisms.Briefly, a variety of general enrichment media are commerciallyavailable and/or can be readily made, these include, but are not limitedto, Tryptone based medium (e.g., Terrific Broth, SOB, SOC, and LBmedium), NZCYM medium, minimal medium, lactose broth, buffered peptonewater, Brain Heart Infusion medium, Haemophilus broth, Tryptic Soybroth, Nutrient broth and the like (see Sambrook et al., MolecularCloning: A Laboratory Manual, 2^(nd) ed., Cold Spring Harbor Press,1989; Ausubel et al., Current Protocols in Molecular Biology, GreenePublishing, 1995; commercially available from Sigma Chemical Co, St.Louis, Mo. and Difco Laboratories Inc., Detroit, Mich.).

[0031] The present invention provides for the detection of targetmicroorganisms which express highly conserved but sterically inaccesibleantigen epitopes by combining an inventive composition of growth mediafollowed by detection with very specific antibodies using a detectionformat, such as a visual immunoprecipitation assay, enzyme linkedimmunoassay, chemiluminescence, immunoblotting, or similar technology.The present invention permits such detection by providing a growthenvironment in a modified culture medium wherein the microorganisms arepermitted to multiply to optimal levels but their surface structure isaltered, without causing substantial cell death, to expose the highlyspecific and conserved antigen epitopes found in the interior cell wallstructure.

[0032] Using the present invention the analyst can incubate the testsample of interest under routine laboratory conditions in the presenceof the inventive growth medium which exposes the specific antigenepitopes. This invention provides a highly accurate test result whilestill affording the analyst with the convenience of standardmicrobiological laboratory conditions. A further aspect of the presentinvention is that no unique or costly equipment and facilities arerequired.

[0033] Since continued cell viability is important to allow the pathogenof interest to grow to sufficient numbers for detection by the chosendetection system (e.g., visual immunoprecipitate assay, enzyme linkedimmunoassay, chemiluminescence, immunoblotting, or similarimmuno-affinity based detection technology), the present inventionutilizes methodologies which simultaneously induce altered cell wallcompositions as well as allowing for further growth of the pathogen.More specifically, the present invention is directed to a highlyspecific detection of target microorganisms by contacting samplespotentially containing these microorganisms in the presence of a growthmedium containing structure modifying organic chemicals which allow theexpression and accessibility of these highly conserved antigen epitopesto specific monoclonal or polyclonal detecting antibodies bound todetecting reagents. Detection is accomplished by means a visualimmunoprecipitate assay, enzyme linked immunoassay, chemiluminescence,immunoblotting, or similar immuno-affinity based detection technology.The present invention permits such detection by modifying the surfacestructure of the target microorganism without causing substantial celldeath in such a manner that the more highly conserved and specificantigen epitopes are made accessible to the corresponding antibodieslinked to detecting reagents.

[0034] The media composition of the present invention biochemicallymodifies the metabolism of the target microorganism so that it producesa modified cell wall which exposes the most specific and conservedepitopes. (See, e.g. Tsang et al., “Screening for Salmonella with aMurine Monoclonal Antibody M105 Detects both Felix O1 BacteriophageSensitive and Resistant Salmonella Strains,” Zbl.Bakt. 286:23-32, 1997;Tsang et al., “A Murine Monoclonal Antibody that Recognizes aGenus-Specific Epitope in the Salmonella Lipopolysaccharide Outer Core,”Zbl.Bakt. 274: 446-455, 1991; Tsang et al., “A Murine MonoclonalAntibody Specific for the Outer Core Oligosaccharide of SalmonellaLipopolysaccharide,” Infection and Immunity, 55: 211-216, 1987; Tsang etal., “Lack of the α-1,2-linked N-acetyl-D-glucosamine epitope in theouter core structures of lipopolysaccharides from the certain Oserogroups and subspecies of Salmonella enterica,” Res. Microbiol. 142:521-533, 1991). The structural modification occurs without inhibitingthe microorganisms ability to grow, therefore, the target pathogenmicroorganism continues to grow uninhibited to reach a detectable level.The combination of structural modification and the ability to furtherreplicate provides an advantage in that the pathogenic microorganismsare generally found in a sample at levels below the detection thresholdof most rapid detection systems. While any detection system may beemployed, preferred detection systems include, but are not limited to,visual immunoprecipitate assay, enzyme linked immunoassay,chemiluminescence, immunoblotting, and similar detection systems.

[0035] In one embodiment of the present invention, a test samplepotentially containing a pathogenic microorganism is contacted with agrowth medium containing an inhibitor of O-antigen polysaccharide cellsurface expression. Subsequently, the sample containing media issubjected to a detection methodology, which may include visualimmunoprecipitate assay, enzyme linked immunoassay, chemiluminescence,immunoblotting, or similar detection systems.

[0036] In preferred embodiments, the compositions, formulae, detectiondevices, and the methods of detecting are specific for Listeria,Enterohemorrhagic E. coli (EHEC), Salmonella, or Campylobacter. In aparticularly preferred embodiment, the inventive growth medium,following incubation, is introduced into a detection system, such as avisual immunoprecipitate assay, an enzyme linked immunoassay,chemiluminescence, immunoblotting, or similar detection technologycontaining an antibody specific for the target microorganism therebyproducing a highly accurate result.

[0037] The present invention combines any of several widely recognizedgeneral enrichment media such as tryptic soy broth, nutrient broth,buffered peptone water, lactose broth, brain heart infusion broth, orsimilar media with a number of antigen structure modifying organicchemicals, including but not limited to 2,4-dinitrophenol, carbonylcyanide-m-chlorophenyl hydrazone or similar electron uncouplers toexpose epitopes which are recognized by monoclonal or polyclonalantibodies which have high affinity for the specific epitopes. Themechanism of action of these organic chemicals is to alter the metabolicpathways of the target microorganism such that it produces a deficientcell wall allowing exposure of the interior specific epitopes. It is thecombination of the alteration of a metabolic pathway which alters thestructure of the cell wall with an antibody-detection reagent containedin a detection device such as visual immunoprecipitate assay which makesthe detection rapid and specific.

[0038] Following incubation in the inventive media under permissiveconditions the results are detected preferably using a rapid detectionmethod such as, but not limited to, visual immunoprecipitate assay,enzyme linked immunoassay, chemiluminescence, immunoblotting, or similardetection technology. Such methodologies are described in greater detailin U.S. Pat. No. 5,658,747 and PCT WO 95/30903. In a preferredembodiment of the invention, the mixture of the composition and the testsample, following incubation, may be exposed to a detergent solution toimprove the accessibility of the conserved antigen epitope. In a mostpreferred embodiment of the invention the detergent may be heated tofacilitate a more rapid exposure of the epitope. In yet anotherembodiment, the mixture, following incubation, is exposed to detergentat an elevated temperature, the temperature is preferably from about 40°C. to 121° C. for a specified time, preferably from two minutes to onehour.

[0039] In one embodiment, the mixture, prior to detection, compromisesup to about 0.02-2.0% by a weight of a detergent, preferably an anionicdetergent, further preferably selected the group consisting of sodiumdodecyl sulfate (SDS) and sodium deoxycholate, and the like, but alsoincluding non-ionic detergents such as NP-40, tergitol and Triton X-100,and the like.

[0040] An additional aspect of the present invention is the use of avisual immunoprecipitate assay to detect the presence of a microorganismin a test sample. In the visual immunoprecipitate assay, the antibodies,including the “antibody-detection-reagent” initially located in thereagent zone, is typically either a polyclonal or monoclonal antibody.Further, when using a polyclonal antibody the antibody is preferablyaffinity column purified prior to its utilization the present invention.The production of such antibodies is well known in the art. (See, e.g.,Antibodies. A Laboratory Manual, Harlow and Lane (eds.), Cold SpringHarbor Laboratory Press, 1988). Suitable affinity purified antibodiescan also be procured from commercially available sources. For example, apolyclonal antisera specific for Salmonella is available from Kirkegaardand Perry Laboratories, Gaithersburg, Md. A preferred visualimmunoprecipitate assay is that which is described by U.S. Pat. No.5,658,747. Briefly, U.S. Pat. No. 5,658,747 utilizes a lateral flowdiagnostic device which comprises a reagent zone containing anantibody-detection reagent and a detection zone located downstream ofthe reagent zone and comprising an immobile binding partner capable ofspecifically binding said complex between the target microorganism andthe antibody detection reagent.

[0041] Polyclonal antibodies can be readily generated by one of ordinaryskill in the art via immunization of a variety of warm-blooded animalssuch as horses, cows, goats, sheep, dogs, chickens, turkeys, rabbits,mice, or rats. Briefly, the target microorganism, or an antigenspecifically associated with the target microorganism, is utilized toimmunize the animal. The immunogenicity of the protein or peptide ofinterest may be increased through the use of an adjuvant such asFreund's complete or incomplete adjuvant or by coupling to anotherprotein such as ovalbumin or keyhole limpet hemocyanin (KLH).

[0042] Monoclonal antibodies can also be readily generated usingwell-known techniques. (See, e.g., Monoclonal Antibodies, Hybridomas: ANew Dimension in Biological Analyses, Plenum Press, Kennett, McKearn,and Bechtol (eds.), 1980, and Antibodies: A Laboratory Manual, Harlowand Lane (eds.), supra.) Briefly, as one example, a subject animal isimmunized as with the production of a polyclonal antibody.Alternatively, in vitro immunization techniques suitable for theproduction of monoclonal antibodies are also known in the art.Antibody-producing cells are then fused to immortal myeloma cells toprovide an immortal hybridoma cell line. Following the fusion, the cellsare placed into culture plates containing a suitable medium,traditionally HAT medium, although other suitable media are known in theart. After about seven days, the resulting fused cells or hybridomas maybe screened in order to determine the presence of antibodies whichrecognize the desired antigen. Following several clonal dilutions andreassays, hybridoma producing antibodies that bind to the protein ofinterest can be isolated.

[0043] Other techniques can also be utilized to construct monoclonalantibodies or binding partners. (See, e.g., Huse et al., “Generation ofa Large Combinational Library of the Immunoglobulin Repertoire in PhageLambda,” Science 246:1275-1281, 1989; Sastry et al., “Cloning of theImmunological Repertoire in Escherichia coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library,” Proc. Natl. Acad. Sci. USA 86:5728-5732,1989; Alting-Mees et al., “Monoclonal Antibody Expression Libraries: ARapid Alternative to Hybridomas,” Strategies in Molecular Biology 3:1-9,1990; Larrick et al., “Polymerase Chain Reaction Using Mixed Primers:Cloning of Human Monoclonal Antibody Variable Region Genes From SingleHybridoma Cells,” BioTechnology 7:934-938, 1989.)

[0044] Once a suitable antibody has been obtained, it may be isolated orpurified by many techniques well known to those of ordinary skill in theart (see Antibodies: A Laboratory Manual, Harlow and Lane, supra).

[0045] Antibodies useful in the present invention are preferably capableof selectively detecting all of the strains of a target microorganism inthe presence of numerous antigenically related organisms. Further, theantibodies are preferably capable of such detection with a low tolerancefor non-specific binding (which leads to a false positive result) and avery low, preferably zero, failure to bind target the microorganism(which leads to a false negative result).

[0046] One aspect of the present invention provides a general enrichmentmedium, tryptic soy broth, containing 0.1-5 mM 2,4-dinitrophenol towhich a test sample is added, thereby forming a mixture, andsubsequently incubated at 37° C. for 6-8 hours. Following incubation, analiquot of the sample is exposed to 0.05-0.5% SDS at 100° C. for tenminutes. The sample may then introduced into a detection device, forexample, a visual immunoprecipitate assay device and observed for theformation of a visual line.

[0047] Preferably, the sample is a solution containing, or consistingessentially of, an unpurified field sample such as a food sample, anenvironmental sample such as water or dirt. Alternatively, the samplemay be a biological fluid such as a body fluid. In a further embodiment,the sample may be partially or substantially purified prior toadministration to the diagnostic device, such as a laboratory sample.Upon contacting the sample with a composition containing a specificantibody-detection reagent for the target microorganism that ispotentially contained within the sample, binding between theantibody-detection reagent and the target microorganism is permitted,thereby detecting the presence or absence of a particular pathogenicmicroorganism.

[0048] Another aspect of the present invention provides a method fordetecting a microorganism in a test sample wherein the test sample isincubated in a general enrichment media comprising at least onestructure modifying organic chemical for sufficient time to propagatedetectable levels of microorganisms. Subsequently, the presence ofpathogenic microorganisms is detected by utilizing immuno-baseddetection methodologies, which include but are not limited to,immuno-affinity, visual immunoprecipitation, enzyme linked immunoassay,chemiluminescence, immunoblotting, and the like. Alternatively, theexposure of antigen in a sample may be enhanced by treatment withdetergent prior to or contemporaneously with detection. In a furtheralternative embodiment, the exposure of antigen in a sample, previouslysubject to propagation in the presence of the composition of the presentinvention, may be enhanced by heating the sample in the presence of thedetergent, prior to or contemporaneously with detection.

[0049] In yet another aspect, the present invention provides methods ofdetecting a target microorganism comprising contacting a samplepotentially containing the target microorganism in the presence of othergenera not of interest but expressing cross reactive antigen epitopeswith a composition as described above under permissive incubationconditions. Following incubation the sample is exposed to an assay suchas the visual immunoprecipitate assay that permits theantibody-detection reagent to bind to the target microorganism toprovide a complex between the target microorganism and theantibody-detection reagent. The complex then migrates downstream alongthe lateral flow membrane to a detection zone containing an immobileantibody capable of binding to the complex to provide a bound complex.Next, the bound complex is detected.

[0050] The following examples are presented for the purpose ofillustration, not limitation.

EXAMPLES Example 1

[0051] Nine strains of Salmonella were identified which werenon-reactive in a visual immunoprecipitate assay. The strains werereported to produce excessive levels of surface antigen. It washypothesized that growth of the strains in the inventive media wouldeliminate or significantly reduce the expression of surface O groupantigen epitopes, thereby allowing detection by highly specificmonoclonal antibodies directed against the core region oflypopolysaccharide contained in the visual immunoprecipitate assaydevice.

[0052] The organisms were grown in the inventive medium, followed byextraction with 0.1% SDS at 100° C. for ten minutes. Strong reactivitywas demonstrated. The inventive media formulation was a commerciallyavailable formulation of tryptic soy broth containing the followingingredients supplemented with 0.5 mM (0.01%) 2,4-dinitrophenol.Trypticase Soy Broth Pancreatic Digest of Casein 17.0 g Papaic Digest ofSoybean Meal 3.0 g Sodium Chloride 5.0 g Dipotassium Phosphate 2.5 gDextrose 2.5 g Distilled Water 1000 ml

Example 2

[0053] Two strains of Salmonella, serogroups A and B, were determined tobe weakly reactive in a monoclonal antibody based visualimmunoprecipitate assay and an enzyme linked immunoassay in the presenceof related competitive microorganisms. These strains were incubated inthe inventive medium in a 1000 fold excess of competitive bacteria, andfound to be highly reactive. The inventive media formulation was acommercially available formulation of buffered peptone watersupplemented with 0.5 mM (0.01%) 2,4-dinitrophenol. Buffered PeptonePancreatic Digest of Gelatin 10.0 g Sodium Chloride 5.0 g DisodiumPhosphate 3.5 g Monopotassium Phosphate 1.5 g Distilled Water 1000 ml

Example 3

[0054] A strain of E. Coli 0157:H7 was found to be weakly reactive in apolyclonal antibody based assay. The strain was grown in the inventivemedium and the sensitivity was improved by 100 fold. The media was amodified tryptic soy broth with 20 mg/ml novobiocin supplemented with0.5 mM (0.01%) 2,4-dinitrophenol. Modified Tryptic Soy Broth BactoTryptone 17.0 g Bacto Soytone 3.0 g Sodium Chloride 5.0 g DipotassiumPhosphate 4.0 g Bile Salts No. 3 1.5 g Bacto Dextrose 2.5 g

Example 4

[0055] A pathogenic strain of Campylobacter jejuni was identified whichdid not react in a polyclonal/monoclonal based enzyme immunoassay. Thestrain was grown in the inventive medium followed by treatment with 0.1%sodium dexoycholate and was found to be strongly reactive. The media wasCampylobacter isolation broth supplemented with 0.5 mM (0.01%)2,4-dinitrophenol. Nutrient Broth No. 2 with 0.6% yeast extractLab-Lemco Powder 10.0 g Peptone 10.0 g Sodium Chloride 5.0 g YeastExtract 6.0 g Distilled Water 1000 ml

[0056] From the foregoing, it will be appreciated that, althoughspecific embodiments of the invention have been described herein forpurposes of illustration, various modifications may be made withoutdeviating from the spirit an scope of the invention. Accordingly, theinvention is not to be limited except as by the appended claims.

We claim:
 1. A composition for exposing antigenic epitopes of amicroorganism, comprising a general enrichment media and at least onestructure modifying organic chemical.
 2. The composition of claim 1,wherein the structure modifying organic chemical is selected from thegroup consisting of 2,4-dinitrophenol and carbonylcyanide-m-chlorophenyl hydrazone.
 3. The composition of either claim 1or 2, wherein the general enrichment media is selected from the groupconsisting of Terrific Broth, SOB medium, SOC medium, LB medium, NZCYMmedium, minimal medium, lactose broth, buffered peptone water, BrainHeart Infusion medium, Haemophilus broth, tryptic soy broth, andnutrient broth.
 4. The composition of claim 3, wherein the structuremodifying organic chemical is 2,4-dinitrophenol.
 5. A method fordetecting a microorganism in a test sample, comprising: (a) contacting atest sample with a composition comprising general enrichment media andat least one structure modifying organic chemical, thereby forming amixture; (b) incubating the mixture for a time sufficient to allow fordetectable levels of microorganisms to develop, and (c) detecting thepresence of specific microorganisms in the mixture.
 6. The methodaccording to claim 5, further comprising contacting the mixture with adetergent, wherein said contact further exposes antigenic epitopes priorto detection.
 7. The method according to claim 6, further comprisingheating the combination of the mixture and detergent prior to detection.8. The method according to either claim 6 or 7, wherein the detergent isan anionic detergent.
 9. The method according to claim 7, wherein thedetergent is selected from the group consisting of sodium dodecylsulfate and sodium deoxycholate.
 10. The method according to claim 6 or7, wherein the detergent is a non-ionic detergent.
 11. The methodaccording to claim 10, wherein the detergent is selected from the groupconsisting of NP-40, tergitol, and triton X-100.
 12. The methodaccording to claim 7, wherein heating is performed at about 40° C. toabout 121° C. for a time sufficient to further expose antigenicepitopes.
 13. The method according to claim 5, wherein the microorganismis selected from the group consisting of Listeria, Enterohemorrhagic E.coli, Salmonella, and Campylobacter.
 14. A method for detecting thepresence of Listeria, Enterohemorrhagic E. coli, Salmonella, orCampylobacter in a test sample, comprising: (a) contacting a test samplewith a composition comprising general enrichment media and at least onestructure modifying organic chemical, thereby forming a mixture; (b)incubating the mixture for a time sufficient to allow for detectablelevels of microorganisms to develop, and (c) detecting the presence ofspecific microorganisms in the mixture, wherein a positive detectionresult indicates the presence of Listeria, Enterohemorrhagic E. coli,Salmonella, or Campylobacter in the test sample.
 15. The methodaccording to claim 14, further comprising contacting the mixture with adetergent, wherein said contact further exposes antigenic epitopes priorto detection.
 16. The method according to claim 15, further comprisingheating the combination of the mixture and detergent prior to detection.17. The method according to claim 15 or 16, wherein the detergent is ananionic detergent.
 18. The method according to claim 16, wherein thedetergent is selected from the group consisting of sodium dodecylsulfate and sodium deoxycholate.
 19. The method according to claim 15 or16, wherein the detergent is a non-ionic detergent.
 20. The methodaccording to claim 19, wherein the detergent is selected from the groupconsisting of NP-40, tergitol, and triton X-100.
 21. The methodaccording to claim 16, wherein heating is performed at about 40° C. toabout 121° C. for a time sufficient to further expose antigenicepitopes.
 22. The method according to claim 14, wherein detection occursby an immunoassay.
 23. The method according to claim 22, wherein theimmunoassay is selected from the group consisting of a visualimmunoprecipitate assay, an enzyme linked immunoassay, chemiluminescence, and immunoblotting.
 24. The method according to claim 23, wherein theimmunoassy is a visual immunoprecipitate assay.
 25. The method accordingto claim 23, wherein the detection utilizes a complementary monoclonalantibody, polyclonal antibody, or an antibody fragment, and wherein saidantibody or antibody fragment is specific for a highly conserved cellwall epitope.
 26. A method for detecting a microorganism in a testsample, comprising contacting a test sample containing a microorganismwith an immunoaffinity based detection device, wherein said test samplehas been previously propagated in the presence of a structure modifyingorganic chemical.
 27. A method for propagating a microorganism such thatcell wall antigen epitopes of the microorganism are altered, comprisingcontacting a test sample with a composition comprising generalenrichment media and at least one structure modifying organic chemicaland propagating the microorganism therein.
 28. A method for detectingmicroorganism specific epitopes on a target microorganism in a testsample, comprising: (a) propagating a microorganism in a test sample ina permissive general enrichment media, wherein said media comprises astructure modifying organic chemical, and (b) contacting the test samplewith a microorganism specific antibody linked to a detecting reagent,wherein reaction with the antibody indicates the presence of themicroorganism.
 29. The method according to claim 28, wherein the contactbetween the test sample and the antibody occurs in device or assaysystem.
 30. The method according to claim 29, wherein the assay systemis selected from the group consisting of a visual immunoprecipitateassay, an enzyme linked immunoassay, chemiluminescence, andimmunoblotting.
 31. The method according to claim 29, wherein the assaydevice is a lateral flow detection device.
 32. The method according toany one of claims 28-31, wherein the antibody is specific for amicroorganism selected from the group consisting of Salmonella,Enterohemorrhagic E. coli, Listeria, and Campylobacter.
 33. A lateralflow device for detecting a target microorganism in a sample comprisinga microorganism specific antibody and a test sample previouslypropagated in a general enrichment media, said media comprising at leastone structure modifying organic chemical.
 34. The device of claim 33,wherein the antibody is specific for Salmonella.
 35. The device of claim33, wherein the antibody is specific for Enterohemorrhagic E. coli. 36.The device of claim 33, wherein the antibody is specific for Listeria.37. The device of claim 33, wherein the antibody is specific forCampylobacter.
 38. The method of any one of claims 5, 14, 26, and 28,wherein said test sample is selected from the group consisting of a foodproduct, water, an environmental sample, a biological sample, a humanspecimen, and a veterinary sample.